The ancient protist, Giardia lamblia colonizes the small intestine of humans and animals. This protozoan parasite lacks mitochondria, highly evolved Golgi structures, and other organelles typical of higher eukaryotes. Nonetheless, it has developed unique metabolic pathways that allow this parasite to survive and multiply in the small intestine by scavenging lipid molecules from the host. Various investigators have shown that Giardia trophozoites are unable to synthesize the majority of their own lipids and cholesterol de novo; rather, they depend mostly on supplies from outside sources. Naturally, the questions of how they scavenge and utilize exogenous lipids for metabolic purposes are extremely important. Earlier we have shown that exogenous phospholipids, once internalized, can be remodeled via deacylation/reacylation and headgroup exchange reactions, which allow Giardia to alter lipids and generate parasite-specific phospho and glycophospholipid molecules. In addition, recent results from our laboratory suggest that transbilayer/ cellular movement of fluorescent-conjugated sphingolipids is dependent on actin filaments and the microtubule network, suggesting the presence of sophisticated lipid transport machinery in Giardia. The major goal of this proposal is to investigate the underlying mechanisms of lipid transport and metabolism in this early-diverging protozoan cell. Therefore, the specific aims are: Aim-1 to investigate the transbilayer lipid transport and trafficking, and the possible role of giardial actin/microtubule cytoskeleton in these processes; Aim-2 to investigate and characterize the regulatory enzyme(s) of phospholipid deacylation/reacylation reactions (i.e., phospholipase/lysophospholipase) by molecular/biochemical methodologies; Aim-3 to study the key enzymes of headgroup exchange pathways, and finally Aim-4 to test the effects of various inhibitors/analogs of lipid metabolic enzymes on in vitro growth of this water-borne pathogen. These studies will yield valuable information regarding lipid synthesis/remodeling by mucosal protozoa, and will lay the foundation for future investigations on novel chemotherapies against Giardia and other related protozoan parasites.